Patient-specific Mutations Research Articles

Abstract 3326: The clinical validity of urinary pellet DNA monitoring of recurrent bladder cancer

Abstract Introduction: Approximately half of high-risk non-muscle-invasive bladder cancers (NMIBCs) recur after transurethral resection of the bladder tumor (TURBT), whereas conventional urological testing for bladder cancer (BC) patients has been considered invasive and with low sensitivity. Our previous reports have shown that circulating tumor DNA (ctDNA) monitored by digital PCR (dPCR) can predict relapse and evaluate treatment efficacy in digestive tract cancers. In BC patients, tumor-derived genetic mutations detected in urinary DNA are expected to be a diagnostic biomarker. This study evaluates the clinical validity of monitoring tumor-specific gene mutations in ctDNA and urine pellet DNA (upDNA) using dPCR as a biomarker for early relapse prediction and determination of treatment efficacy. Materials and Methods: Thirty-two previously treated and untreated BC patients were enrolled. Tumor DNA was extracted from archived TURBT tissues, and gene mutation analysis by Next Generation Sequencing (NGS) of tumors was performed to select patient-unique somatic mutations for both ctDNA and upDNA monitoring by dPCR. Longitudinal variant allele frequencies (VAFs) of ctDNA and upDNA were monitored by dPCR for up to two years. While dPCR is rapid and has a high sensitivity of less than 0.1% VAF, a mutation-specific primer/probe set is required. Therefore, we established over 1,000 primer/probe sets for frequent mutations in human cancer. This large-scale dPCR primer/probe library allow us to select patient specific mutations for immediate ctDNA monitoring. Clinical recurrence (Crec) based on imaging and urinary cell findings are compared with VAF dynamics of ctDNA and upDNA. Results: The median observation period was 516 days (30-733), and a total of 230 urine samples were collected. The selection of monitoring mutations based on NGS analysis was performed in 93.8% (30/32) cases, with 2.3 (range: 1-6) mutations monitored per case. The primer/probe sets covered 87.5% (28/32) of cases and 87.5% (42/48) of mutations. The VAFs of the upDNA of cases started showing constantly increasing trends 3-12 months earlier than the diagnosis of Crec in 71.4% (5/7) of the cases. The other two Crec cases, which did not show an elevated upVAF, were both pyuria cases. After local treatment, the upDNA VAFs remained high in the Crec cases. Conventional Crec did not seem to be fully reflected by ctDNA alone. Conclusion: Frequent VAF monitoring using upDNA by dPCR enables the prediction of local relapse and the evaluation of treatment efficacy in the management of BC patients. Citation Format: Masakazu Abe, Hayato Hiraki, Takashi Tsuyukubo, Sadahide Ono, Shigekatsu Maekawa, Daichi Tamura, Daiki Ikarashi, Renpei Kato, Tomohiko Matsuura, Mitsugu Kanehira, Ryo Takata, Hiromitsu Fujisawa, Takeshi Iwaya, Masashi Idogawa, Wataru Obara, Satoshi S. Nishizuka. The clinical validity of urinary pellet DNA monitoring of recurrent bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3326.

375 Vorinostat for Missense Mutated Von Hippel Lindau Disease Associated Hemangioblastomas: A Pilot Study

INTRODUCTION: Missense mutated VHL protein (pVHL) maintains intrinsic function but undergoes proteasomal degradation, loss of function and tumor initiation/progression of VHL-associated tumors. Histone deacetylase inhibitors (HDACi) can rescue missense mutated pVHL and arrest tumor growth in pre-clinical models. METHODS: Seven subjects with missense VHL received 400mg/day of vorinostat (7-days) and surgical resection of hemangioblastomas. We analyzed vorinostat-treated HBs (and paired untreated HBs from the same patients) for gene and protein expression in the pVHL-HIF-VEGF pathway, and protein-protein interactions. We explored the transcriptional landscape of these tumors with single nucleus gene expression analysis. We recapitulated patient specific missense mutations and validated the clinical findings using lentiviral expression recombinant constructs in VHL-/- 786-O cells. RESULTS: Short-term oral vorinostat was well tolerated by all patients (age 46.0 ± 14.5 years; 25-61 years). In the 6 evaluable tumors, pVHL expression in neoplastic stromal cells of vorinostat treated tumors was significantly elevated compared to untreated tumors (p = 0.01). We found transcriptional suppression of downstream effectors including VEGF-A and GLUT1 in human tumor samples and validated the effects in patient-specific missense mutations expressed in VHL 786-O cells. Mechanistically, we found that vorinostat prevented Hsp90 recruitment to missense mutated pVHL. The vorinostat effects on pVHL rescue, transcriptional repression and Hsp90-pVHL interaction was independent of the location of the mutation on VHL. Lastly, we found a HB stromal cell specific effect in suppression of pro-tumorigenic pathways with single nucleus gene expression analysis. CONCLUSIONS: Short-term treatment with vorinostat increased pVHL levels and suppressed tumor progression signaling in VHL-associated hemangioblastomas. These results indicate that proteostasis modulation can rescue tumor pVHL in germline missense mutated VHL patients and such treatment could arrest tumor growth.

Persistent Molecular Disease in Adult Patients With AML Evaluated With Whole-Exome and Targeted Error-Corrected DNA Sequencing.

Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML. The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq). WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A, ASXL1, and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy. PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

Ultrasensitive Circulating Tumor DNA Pilot Study Distinguishes Complete Response and Partial Response With Immunotherapy in Patients With Metastatic Renal Cell Carcinoma.

Circulating tumor DNA (ctDNA) has been validated across multiple indications in the adjuvant and surveillance settings. We evaluated whether targeted digital sequencing (TARDIS) may distinguish a partial response (PR) from a complete response (CR) among patients with metastatic renal cell carcinoma (mRCC) receiving immune checkpoint inhibitor (ICI) therapy. Eligible patients had mRCC that yielded a PR or CR to ICI therapy. Peripheral blood was obtained at a single time point for ctDNA analysis. TARDIS was used for quantification of average variant allele fractions (VAFs). Our primary objective was to determine the association between VAFs and depth of response (PR v CR). A secondary objective was to determine whether VAFs were associated with disease progression. Twelve patients were analyzed, nine of whom achieved a PR (75%). Patients received either nivolumab monotherapy (50%) or nivolumab plus ipilimumab (50%). ctDNA analysis incorporated an average of 30 patient-specific mutations (range, 19-35); average coverage depth was 103,342 reads per target. TARDIS quantified a significant difference in VAFs between PR and CR (median, 0.181% [IQR, 0.077%-0.420%] v 0.007% [IQR, 0.0%-0.028%], respectively [P = .014]). Of the 12 patients in the series, six patients demonstrated radiographic progression subsequent to ctDNA assessment. Patients who progressed on subsequent scans had significantly higher ctDNA than those who maintained their response (median, 0.362% [IQR, 0.181%-2.71%] v 0.033% [IQR, 0.007%-0.077%], respectively [P = .026]). In this pilot study, TARDIS accurately differentiated PR from CR among patients with mRCC receiving immunotherapy, and also prospectively identified patients at risk for subsequent progression. Given these findings, we envision subsequent studies that validate these results and investigate the utility of this assay to discern appropriate candidates for discontinuation of immunotherapy.

Retinal Organoids from an AIPL1 CRISPR/Cas9 Knockout Cell Line Successfully Recapitulate the Molecular Features of LCA4 Disease.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is expressed in photoreceptors where it facilitates the assembly of phosphodiesterase 6 (PDE6) which hydrolyses cGMP within the phototransduction cascade. Genetic variations in AIPL1 cause type 4 Leber congenital amaurosis (LCA4), which presents as rapid loss of vision in early childhood. Limited in vitro LCA4 models are available, and these rely on patient-derived cells harbouring patient-specific AIPL1 mutations. While valuable, the use and scalability of individual patient-derived LCA4 models may be limited by ethical considerations, access to patient samples and prohibitive costs. To model the functional consequences of patient-independent AIPL1 mutations, CRISPR/Cas9 was implemented to produce an isogenic induced pluripotent stem cell line harbouring a frameshift mutation in the first exon of AIPL1. Retinal organoids were generated using these cells, which retained AIPL1 gene transcription, but AIPL1 protein was undetectable. AIPL1 knockout resulted in a decrease in rod photoreceptor-specific PDE6α and β, and increased cGMP levels, suggesting downstream dysregulation of the phototransduction cascade. The retinal model described here provides a novel platform to assess functional consequences of AIPL1 silencing and measure the rescue of molecular features by potential therapeutic approaches targeting mutation-independent pathogenesis.

Abstract P5-05-01: Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer

Abstract Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF< 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-01.

Establishment of iPS cell line (KLRMMEi003-A) from a patient with Usher syndrome due to USH2A mutation

We generated an induced pluripotent stem (iPS) cell line by reprogramming peripheral blood mononuclear cells of a patient with Usher syndrome type II carrying USH2A gene mutation (c.8559-2A > G). The iPS cell line with confirmed patient-specific point mutation exhibited typical iPS cell characteristics and maintained a normal karyotype. It can be used as 2D and 3D models to investigate the underlying pathogenic mechanism and lay a solid foundation for future personalized therapy.

Efficient detection and monitoring of pediatric brain malignancies with liquid biopsy based on patient-specific somatic mutation screening.

Brain cancer is the leading cause of cancer-related death in children. Early detection and serial monitoring are essential for better therapeutic outcomes. Liquid biopsy has recently emerged as a promising approach for detecting these tumors by screening body fluids for the presence of circulating tumor DNA (ctDNA). Here we tested the limits of liquid biopsy using patient-specific somatic mutations to detect and monitor primary and metastatic pediatric brain cancer. Somatic mutations were identified in 3 ependymoma, 1 embryonal tumor with multilayered rosettes, 1 central nervous system neuroblastoma, and 7 medulloblastoma patients. The mutations were used as liquid biomarkers for serial assessment of cerebrospinal fluid (CSF) samples using a droplet digital PCR (ddPCR) system. The findings were correlated to the imaging data and clinical assessment to evaluate the utility of the approach for clinical translation. We developed personalized somatic mutation ddPCR assays which we show are highly specific, sensitive, and efficient in detection and monitoring of ctDNA, with a positive correlation between presence of ctDNA, disease course, and clinical outcomes in the majority of patients. We demonstrate the feasibility and clinical utility of personalized mutation-based liquid biopsy for the surveillance of brain cancer in children. However, even with this specific and sensitive approach, we identified some potential false negative analyses. Overall, our results indicate that changes in ctDNA profiles over time demonstrate the great potential of our specific approach for predicting tumor progression, burden, and response to treatment.

Evolving Applications of Circulating Tumor DNA in Merkel Cell Carcinoma.

Circulating tumor DNA (ctDNA) is a subset of circulating cell-free DNA released by lysed tumor cells that can be characterized by its shorter strand length and tumor genome-specific information. The relatively short half-life of ctDNA allows it to provide a real-time measure of tumor burden which has potential prognostic and surveillance value as a tumor biomarker. Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer that requires close monitoring due to the high risk of relapse. There are currently no good tumor biomarkers for MCC patients, especially those who are negative for Merkel cell polyomavirus. ctDNA shows promise for improving the prognoses of MCC patients by monitoring tumor burden, identifying minimal residual disease (MRD), and stratifying patients by their likelihood of response to immune checkpoint inhibition or risk of relapse. In particular, bespoke ultra-sequencing platforms allow for the creation of patient-specific mutation panels that improve ctDNA detection, especially for patients with rare or uncharacteristic mutations. Leveraging bespoke ctDNA assays may improve physicians' ability to alter treatment plans for non-responsive or high-risk patients. In addition, ctDNA MRD monitoring may allow physicians to treat relapses early before clinically evident disease is present.

Human MD2 deficiency—an inborn error of immunity with pleiotropic features

Toll-like receptors (TLRs) are important pattern recognition receptors that sense microbes and control host defense. Myeloid differentiation protein 2 (MD2) is the indispensable coreceptor for TLR4, facilitating the binding to the gram-negative bacterial cell wall component LPS and activation of downstream signaling. We sought to provide phenotypic and mechanistic insights into human MD2 deficiency. To elucidate the genetic cause in a patient with very early onset inflammatory bowel disease, we performed whole-exome sequencing and studied the functional consequences of the identified mutation in LY96 (encoding for MD2) in genetically engineered induced pluripotent stem cell-derived macrophages with knockout of MD2 or knockin of the patient-specific mutation, including TLR4-mediated signaling, cytokine production, and bacterial handling. Whole-exome sequencing identified a homozygous in-frame deletion in the LY96 gene (c.347_349delCAA; p.Thr116del) in a patient with very early onset inflammatory bowel disease and a sibling presenting with pneumonia and otitis media. Induced pluripotent stem cell-derived macrophages with knockout of MD2 or expression of the Thr116del mutation showed impaired activation of nuclear factor kappa B and mitogen-activated protein kinase signaling as well as TLR4 endocytosis on challenge with LPS or bacteria. In addition, MD2-deficient macrophages showed decreased cytokine expression (eg, IL-6, TNF, and IL-10) in response to LPS or gram-negative but not gram-positive bacteria. Human MD2 deficiency causes defective TLR4 signaling in response to LPS or gram-negative bacteria. The clinical manifestations and expressivity might be variable due to unknown secondary risk factors. Because TLR4 represents a therapeutic target for multiple inflammatory conditions, our study may provide insights into potential side effects of pharmacological TLR4 targeting.

Detecting the Unusual without Compromising Diagnostic Accuracy - a Prospective WGS/Wts Pilot Study in Acute Leukemias Provided Additional Information for Diagnosis, Prognosis and Treatment

Background: Following WHO and ELN guidelines, standard diagnostics and risk stratification for acute leukemias (AML and ALL) are based on genetic information gathered by a combination of cytogenetics and targeted molecular profiling. Today, comprehensive techniques, i.e WGS and WTS (whole genome and transcriptome sequencing) and the accompanying clinical bioinformatics workflows may even refine and individualize diagnostics. Aim: To evaluate the clinical utility and added value of WGTS in a real-world setting in comparison to standard of care genetic diagnostics. Patients and Methods: The study enrolled 113 patients, 97 with AML and 16 with ALL, diagnosed according to WHO criteria based on cytomorphology, immunophenotyping, cyto- and molecular genetics. WGS (100x) and WTS (50 Mio reads) were performed on NovaSeq instruments. For selected AML cases CD3+ MAC sorted cells were used as normal controls (30x). Variants were called with Strelka2, Manta and GATK using a tumor w/o normal pipeline, fusions with Arriba, STAR-Fusion and Manta. Results: All patients were analyzed for structural variants (SV), copy number variations (CNV) and copy-neutral loss of heterozygosity (cnLOH) based on WGS data and the presence of fusion transcripts and expression patterns based on WTS data. Samples without a normal control (n = 74) were analyzed for 121 genes recurrently mutated in myeloid/lymphoid neoplasms, whereas for 39 AML cases with CD3+ normal controls the whole exome was assessed. Overall, 308/333 (92%) gene mutations were consistently identified by targeted sequencing and WGS. Due to the higher read depth, 25/333 (8%) sub-clonal mutations were only detected by targeted sequencing, with 23/25 showing a variant allele fraction <10%. However, 40 gene mutations were identified exclusively by WGS due to the unrestricted analytical scope (e.g. PTPN11, DDX54). Every patient carried on average 3.3 gene mutations within the 121 genes of interest. The number increased to 63 variants (mean; range: 46-116) when analyzing the exome. 52/63 variants are most likely germline variants based on occurrence in CD3+ control sample. Hence, the CD3+ control samples reduced the number of somatic variants in the exome to 11 (mean; range: 1-23) mostly patient-specific mutations that can be explored for personalized treatment options. Addressing cytogenetics with relevance to risk stratification and assignment to AML/ALL genetic subgroups, WTS identified all entity defining fusions and WGS and chromosome banding analysis (CBA) showed a very high concordance. In 10 patients WGS missed subclonal aberrations due to small clone size (median clone size detected by IP-FISH analysis: 6%). However, WGS provided additional information in 40 patients, mostly SV, CNV and cnLOH of unknown significance, but also alterations of biological interest and prognostic and/or therapeutic importance: in one AML case WGTS revealed a KMT2A::MLLT10 rearrangement that was cytogenetically cryptic and not detectable by FISH (KMT2A break apart probe), assigning the patient to the adverse risk group. WGS also identified a cytogenetically cryptic SV leading to an ETV6::ABL1 rearrangement in an ALL patient with complex karyotype. Retrospective analysis showed that the aberration had already been present when the patient presented with an MPN and a normal karyotype (CBA) two years earlier. Hence, WGS led to the diagnosis of an ETV6::ABL1-positive neoplasm in blast phase (poor prognosis), which might be treated with tyrosine kinase inhibitors. Of note, in 3 AML patients a 7q deletion or complex SV leading to a 7q deletion resulted in a CDK6::NOM1 fusion transcript and high MNX1 expression. In addition, WGS revealed in one patient with ETP-ALL a cytogenetically cryptic translocation t(7;14)(q36;q32) affecting the IGH locus and the NOM1 gene, accompanied by high MNX1 expression. Elevated MNX1 expression as a result of t(7;12)(q36;p13) is a very rare event in pediatric AML, but our findings might suggest a more important role of MNX1 expression in adult acute leukemia than previously known. Conclusions: This prospective study in a real-world setting 1) demonstrated the diagnostic power of WGTS with an accuracy comparable to gold standard techniques, 2) added important prognostic information, 3) identified potential new therapeutic targets and 4) gave new insights into disease biology. WGTS significantly will improve and refine diagnostics in the future.

Proteogenomic Analysis of SF3B1mut CLL Reveals Altered Protein Signaling Cascades and Metabolomic Pathways

Although mutation in the U2 snRNP spliceosome component SF3B1 has been appreciated as a common driver of chronic lymphocytic leukemia (CLL) for over a decade, we still know little of its mechanism of action. Prior transcriptome-based characterizations have demonstrated widespread changes in alternative splicing in SF3B1mut CLL. We hypothesized that protein level characterization could potentially yield fresh insights on alternative expression of proteins and on the impact of altered signaling pathways in patients bearing this mutation. To this end, we assembled CLL specimens from a cohort of 105 patients enrolled on the German CLL Study Group (GCLLSG) trials CLL8 and CLL11, collected prior to therapy. Of these, 52 patients expressed SF3B1wt and 53 patients harbored clonal SF3B1mut (CCF>0.8); 25 of these were the K700E mutation. Additionally, we characterized these samples by WES (available for 97 patients) and RNA-Seq (available for 96 patients). Proteomes and phosphoproteomes from all 105 samples were analyzed using a multiplexed isobaric-tagged (TMT) high resolution mass spectrometry (MS) workflow. Briefly, cells were lysed and proteins were digested into peptides with LysC and trypsin. Protein yield per sample ranged from 20 μg to 300 μg. Peptides were labeled with TMT-11 reagent and combined into 11 multiplexes while accounting for input material availability and randomizing for SF3B1 mutation, IGHV status and ATM/TP53 mutation. To enable cross TMT-plex comparison, we constructed a common reference consisting of 40 samples with highest protein amount and an equal representation between SF3B1wt and SF3B1mut samples. Each plex was fractionated into 24 fractions by high pH reversed phase separation and 500 ng protein per fraction were analyzed by MS (Orbitrap Exploris, Thermo Fisher Scientific). For phosphoproteome analysis, peptides were pooled into 12 fractions and enriched by IMAC prior to MS analysis. Spectra were analyzed by Spectrum Mill v7.1 (Broad Institute) and searched against a human reference proteome database supplemented with non-canonical ORFs supported by Riboseq evidence of translation (nuORFdb v1.0). Patient specific proteins containing single amino variants, indels, and splice isoforms, derived from WES and RNA-Seq, were included in the search database. We obtained a total of 10,419 proteins, with a median of 8,882 proteins per TMT-plex and 6,969 proteins that were quantifiable across all samples. The phosphoproteome analysis identified 32,410 sites; while each TMT-plex yielded a median of 19,380 phosphosites per plex, 5,810 were quantifiable across all patients. We integrated proteome data with patient specific mutation calls, RNA-Seq and copy number alteration (CNA) data using Panoply, a cloud-based platform for proteogenomic data analysis (Broad Institute). We confirmed the impact of chr11 and chr13 deletion as well as chr12 amplification on protein expression. We readily identified peptides spanning the SF3B1mut amino acid regions in corresponding samples. By unsupervised clustering, samples were grouped by SF3B1 status on the CNA and proteome level, a trend not observed based on RNA or phosphoproteome analysis. Gene set enrichment analysis revealed enrichment of TNFA and PI3K signaling in the SF3B1wt clusters, while the SF3B1mut associated clusters showed a strong association with metabolism changes. This analysis was highly concordant with a parallel interrogation comparing proteins expressed in SF3B1wt CLL versus SF3B1mut CLL. Here 1,955 proteins and 1,737 phosphosites were significantly altered (5% FDR). Proteins involved in B cell receptor and MAP kinase signaling were highly expressed in SF3B1wt samples, while again amino acid metabolism pathways as well as oxidative phosphorylation were upregulated in SF3B1mut samples. To more deeply investigate the role of metabolism pathways in SF3B1mut CLL, we subjected 6 independent CLL samples to MS-based metabolomics analysis. Inosine (P=0.029) and guanosine (P=0.047) were lower in the SF3B1mut extracts (P=0.001), while acylcarnitines (P=0.0004) were increased, indicating fatty acid oxidation as a preferred energy source in SF3B1mut CLL. Altogether, our study reveals fresh evidence of altered energy metabolism in SF3B1mut CLL. Ongoing studies focus on defining the mechanism by which SF3B1mut exerts these metabolic changes and novel therapeutic opportunities afforded by these findings.

The Rise and Fall of Leukemia Clones in Longitudinal Samples from Diagnosis to Relapse in KMT2A-rearranged Infant and Childhood Leukemia

The genomic landscape, evolutionary trajectories and the mechanisms driving relapse in KMT2A-rearranged (KMT2A-r) infant and childhood acute lymphoblastic (ALL) and acute myeloid leukemia (AML) are not completely understood. We therefore studied 30 KMT2A-r ALL (n=16) and AML (n=14) patients of which 19 relapsed. Eleven diagnose-relapse-germline trios were analyzed by whole genome (WGS) and whole exome sequencing (WES), and all patients by ultra-deep resequencing (average 3400X) of patient-specific mutations identified by WGS/WES on the diagnostic (n=30) and relapse (n=11) samples, at diagnosis (n=30), during treatment (n=203) and at relapse (n=30, 1-4 bone marrow (BM) relapses in 17 patients). The mutational burden increased from diagnosis to relapse in the diagnose-relapse-germline trios. The combined analysis of WGS/WES and ultra-deep data showed that the leukemias typically evolved through branching evolution, and that a selective sweep occurred at first relapse in 76% of patient. Further, additional subclones were often detected at relapse and in 69% the relapse clone was detected already at diagnosis as a subclone. Eight pathways were recurrently altered including signalling, cell cycle, epigenetic, B-cell development, transcription factor, glucocorticoid receptor signalling, purine metabolism and cohesin. Signalling mutations were the most common type of mutations at diagnosis (50%) and at relapse (41%) and the diagnostic frequency was similar in patients that remained in complete remission (CR) and in those that relapsed (55% versus 47%). Signalling mutations were often subclonal (60%) and 30% had more than one mutation and mutations in signalling genes were usually gained or maintained at relapse in ALL but mainly lost or reduced in size in AML. Alterations in cell cycle genes were enriched at relapse and either gained (60%) or maintained (27%), and included TP53 in ALL and AML, CDKN2A/B in ALL and CCND3 in AML. The TP53 alterations were typically biallelic at relapse. Only 2/30 patients had diagnostic TP53 mutations and both relapsed. Further, relapse-specific mutations in genes involved in glucocorticoid signalling and purine metabolism (CREBBP, NT5C2,PRPS2, NR3C1) were detected in all ALL patients with early relapse (n=4, during maintenance therapy). These relapse samples also had co-occurring IKZF1 deletions with 3 acquired at relapse and 1 maintained, as well as acquired TP53 mutations (3/4 patients). By contrast, infant ALL with a very early relapse (n=4, before maintenance therapy) lacked cell cycle and chemoresistance-associated mutations at relapse. Ultra-deep sequencing did not detect the CREBBP, NT5C2, PRPS2 or TP53 mutations at diagnosis and manual inspection of the WGS reads failed to detect the NR3C1 deletion. In AML, gain of WT1 mutations was seen in patients with late relapse (after >1 year in CR1). Longitudinal sequencing on the 30 patients showed that at day 15, ALL patients that remained in CR (n=5) had higher levels of molecularly detectable leukemia cells as compared to those that relapsed (n=8), however at day 29, the reverse was seen with relapse patients having higher levels (n.s). Further, infants with very early relapse (n=3) had a higher leukemic burden at both time points as compared to those with early relapse (n=5). Patients with very early relapse typically reached CR later and the relapse clone could be detected at diagnosis in all 4 patients compared to 2/4 with early relapse. In 7 patients, 4 that relapsed and 3 that remained in CR, low-frequency mutations and KMT2A-fusion positive leukemic clones (DNA-level) were found at remission. Moreover, 4 patients had molecularly detectable clones across all investigated time points until relapse (6-13 time points), 2 had persistent disease but 2 entered CR. Finally, the relapse clone could be detected 46-126 days before relapse in 6 patients. Combined, early relapse in infant and childhood KMT2A-r ALL was characterized by relapse-specific TP53, IKZF1, CREBBP, NR3C1, NTC52 or PRPS2 alterations. By contrast, very early relapse KMT2A-r infant ALL likely arises from pre-existing chemo resistant cells with a paucity of genetic alterations in relapse and chemoresistance-associated genes at relapse. Further, longitudinal molecular tracking provided biological insight into clonal response to treatment and captured the complexity of the temporal evolution in very early and early relapse patients.

Temporal dynamics of RSV shedding and genetic diversity in adults during the COVID-19 pandemic in a French hospital, early 2021

Human respiratory syncytial virus (RSV) is responsible of lower respiratory tract infections which may be severe in infants, elderly and immunocompromised adults. Europe and North-American countries have observed a massive reduction of RSV incidence during the 2020–2021 winter season. Using a systematic RSV detection coupled to SARS-CoV-2 for all adult patients admitted at the Foch hospital (Suresnes, France) between January and March 2021 (n = 11,324), only eight RSV infections in patients with prolonged RNA shedding were diagnosed. RSV whole-genome sequencing revealed that six and two patients were infected by RSV groups A and B, respectively. RSV carriage lasted from 7 to at least 30 days disregarding of RSV lineage. The most prolonged RSV shedding was observed in an asymptomatic patient. We detected novel patient-specific non-synonymous mutations in the G glycoprotein gene, including a double identical mutation in the repeated region for one patient. No additional mutation occurred in the RSV genome over the course of infection in the four patients tested for. In conclusion, our results suggest that the temporal shift in the RSV epidemic is not likely to be explained by the emergence of a high frequency, unreported variant. Moreover, prolonged RSV carriages in asymptomatic patients could play a role in virus spread.

Development of a Merkel polyomavirus-specific T cell receptor analysis tool to guide cancer immunotherapy

Background and aims: It is difficult to identify cancer-specific T cells in most malignancies as they are driven by patient-specific mutations. However, 80% of Merkel cell carcinoma (MCC) cases are driven by a polyomavirus (MCPyV). Importantly, the T cell immune response is focused on the short, almost invariant viral oncoproteins. Virus-driven MCC thus provides an opportunity to quantitatively characterize tumor-specific T cells. We developed specific tetramer reagents that bind T cell receptors (TCR) to identify tumor-reactive T cells.

Shared and Distinct Functional Effects of Patient-Specific Tbr1 Mutations on Cortical Development.

T-Box Brain Transcription Factor 1 (TBR1) plays essential roles in brain development, mediating neuronal migration, fate specification, and axon tract formation. While heterozygous loss-of-function and missense TBR1 mutations are associated with neurodevelopmental conditions, the effects of these heterogeneous mutations on brain development have yet to be fully explored. We characterized multiple mouse lines carrying Tbr1 mutations differing by type and exonic location, including the previously generated Tbr1 exon 2-3 knock-out (KO) line, and we analyzed male and female mice at neonatal and adult stages. The frameshift patient mutation A136PfsX80 (A136fs) caused reduced TBR1 protein in cortex similar to Tbr1 KO, while the missense patient mutation K228E caused significant TBR1 upregulation. Analysis of cortical layer formation found similar defects between KO and A136fs homozygotes in their CUX1+ and CTIP2+ layer positions, while K228E homozygosity produced layering defects distinct from these mutants. Meanwhile, the examination of cortical apoptosis found extensive cell death in KO homozygotes but limited cell death in A136fs or K228E homozygotes. Despite their discordant cortical phenotypes, these Tbr1 mutations produced several congruent phenotypes, including anterior commissure reduction in heterozygotes, which was previously observed in humans with TBR1 mutations. These results indicate that patient-specific Tbr1 mutant mice will be valuable translational models for pinpointing shared and distinct etiologies among patients with TBR1-related developmental conditions.SIGNIFICANCE STATEMENT Mutations of the TBR1 gene increase the likelihood of neurodevelopmental conditions such as intellectual disability and autism. Therefore, the study of TBR1 can offer insights into the biological mechanisms underlying these conditions, which affect millions worldwide. To improve the modeling of TBR1-related conditions over current Tbr1 knock-out mice, we created mouse lines carrying Tbr1 mutations identical to those found in human patients. Mice with one mutant Tbr1 copy show reduced amygdalar connections regardless of mutation type, suggesting a core biomarker for TBR1-related disorders. In mice with two mutant Tbr1 copies, brain phenotypes diverge by mutation type, suggesting differences in Tbr1 gene functionality in different patients. These mouse models will serve as valuable tools for understanding genotype-phenotype relationships among patients with neurodevelopmental conditions.

Abstract P2107: Improving Stem Cell Models Of Duchenne Muscular Dystrophy-related Cardiomyopathy By Incorporation Of Physiologic Mechanical Stress

Background: Cardiomyopathy in Duchenne muscular dystrophy (DMD) is a significant driver of disease morbidity and mortality. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study patient-specific mutations in the dish, however they generally suffer from immaturity. DMD is caused by mutations in the gene DMD , which codes for the protein dystrophin. Dystrophin localizes to the sarcolemma, forming part of a critical transmembrane mechanical and signaling complex that bridges the sarcomere to the extracellular matrix. Disruption of this axis leads to membrane fragility and mechanical stress is a main driver of DMD pathology. New therapies for DMD are in clinical development, including exon skipping, micro-dystrophin gene therapy, CRISPR-based gene editing, and membrane resealants, with some exon skipping strategies receiving FDA approval. The main outcomes of these therapies have been tied to improvement in skeletal muscle function with cardiac efficacy remaining understudied, despite its clear importance for patient outcomes. Results: Equibiaxial strain was employed as a physiologic mechanical stressor to simulate the in vivo mechanism of myocyte injury in an in vitro format using patient-derived DMD iPSC-CMs. By titrating strain, we determined that application of 10% strain for 2 hours robustly differentiates DMD and control iPSC-CMs using the clinically relevant biomarker LDH release as a proxy for membrane damage (control LDH release fold change: no significant change; DMD LDH release fold change: 2.51, p&lt;0.0001). We evaluated DMD biomarkers released into the media using aptamer-based proteomics profiling and identified a striking strong correlation with serum biomarkers from DMD patients. Longer duration strain (24 hours) led to cell injury in both control and DMD iPSC-CMs. A membrane resealant was evaluated in this platform, which demonstrated enhanced recovery following treatment for both DMD and control iPSC-CMs with concomitant reduction in biomarker release. Conclusions: iPSC-CM models can be improved by incorporating physiologic mechanical stress, which is a main driver pathology. This platform is especially useful for evaluating dystrophic cardiomyopathy therapeutics.

Personalized Medicine Approach in a DCM Patient with LMNA Mutation Reveals Dysregulation of mTOR Signaling.

Background: Mutations in the Lamin A/C (LMNA) gene are responsible for about 6% of all familial dilated cardiomyopathy (DCM) cases which tend to present at a young age and follow a fulminant course. Methods: We report a 47-year-old DCM patient with severely impaired left ventricular ejection fraction and NYHA functional class IV despite optimal heart failure treatment. Whole-exome sequencing revealed an LMNA E161K missense mutation as the pathogenetic cause for DCM in this patient. We generated a patient-specific LMNA-knock in (LMNA-KI) in vitro model using mES cells. Results: Beta adrenergic stimulation of cardiomyocytes derived from LMNA-KI mES cells resulted in augmented mTOR signaling and increased dysregulation of action potentials, which could be effectively prevented by the mTOR-inhibitor rapamycin. A cardiac biopsy confirmed strong activation of the mTOR-signaling pathway in the patient. An off-label treatment with oral rapamycin was initiated and resulted in an improvement in left ventricular ejection fraction (27.8% to 44.5%), NT-BNP (8120 ng/L to 2210 ng/L) and NYHA functional class. Conclusion: We have successfully generated the first in vitro model to recapitulate a patient-specific LMNA E161K mutation which leads to a severe form of DCM. The model may serve as a template for individualized and specific treatment of heart failure.

Interrogating breast cancer heterogeneity using single and pooled circulating tumor cell analysis

Single cell technologies allow the interrogation of tumor heterogeneity, providing insights into tumor evolution and treatment resistance. To better understand whether circulating tumor cells (CTCs) could complement metastatic biopsies for tumor genomic profiling, we characterized 11 single CTCs and 10 pooled CTC samples at the mutational and copy number aberration (CNA) levels, and compared these results with matched synchronous tumor biopsies from 3 metastatic breast cancer patients with triple-negative (TNBC), HER2-positive and estrogen receptor-positive (ER+) tumors. Similar CNA profiles and the same patient-specific driver mutations were found in bulk tissue and CTCs for the HER2-positive and TNBC tumors, whereas different CNA profiles and driver mutations were identified for the ER+ tumor, which presented two distinct clones in CTCs defined by mutations in ESR1 Y537N and TP53, respectively. Furthermore, de novo mutational signatures derived from CTCs described patient-specific biological processes. These data suggest that tumor tissue and CTCs provide complementary clinically relevant information to map tumor heterogeneity and tumor evolution.

S167: PREDICTION OF RELAPSE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION USING INDIVIDUALIZED MEASURABLE RESIDUAL DISEASE MARKERS; THE PROSPECTIVE NORDIC STUDY NMDSG14B

Background: One third of patients with myelodysplastic syndrome (MDS) relapse after allogeneic stem cell transplantation (HCT). Early detection of impending relapse would enable pre-emptive treatment and potentially reduce relapse risk but is limited by the lack of sensitive markers for measurable residual disease (MRD). We developed a pipeline where patient-specific mutations, as determined by a myeloid next generation sequencing (NGS) panel are tracked using digital droplet PCR (ddPCR). Aims: To evaluate if personalized MRD detection by ddPCR can predict clinical relapse earlier than conventional methods. Methods: The prospective study (NCT02872662) enrolled patients with MDS, MDS/MPN or MDS-AML with < 30% marrow blasts undergoing HCT. Patients were included before HCT, and serial bone marrow (BM) samples were collected every third month post-HCT for 2 years. Peripheral blood (PB) samples were collected monthly. MRD results were not available for the treating physician. Results: We screened 286 pts between 2016 and 2020, whereof 20 were excluded mainly due to lack of genetic aberration or no HCT performed. 266 pts were included from 12 HCT centers. Median age was 64 (18-78) years and 59% were male. Myeloid panel NGS screening identified a median of 2 (0-9) mutations. The most common mutations were TET2 (n=85), ASXL1 (n=73) and SRSF2 (n=59). Median time of follow up was 886 (4-1934) days. Sixty pts relapsed after a median of 189 (53-1281) days and 46 died due to non-relapse mortality after a median of 121 (4-1036) days. Remaining pts (n=160) were in continuous complete remission (CCR) after a median follow-up of 1053 (479-1934) days. Estimated 1 and 2y overall survival was 79%, and 71%, respectively, while estimated 1 and 2y relapse-free survival (RFS) was 75% and 66%, respectively. MRD data was missing in 46 pts; no post-HCT samples available (n=15), no mutation detected (n=14) and difficulties to design ddPCR primers (n=11). 221 pts were available for MRD analysis with a median number of 4 (0-13) and 5 (0-23) samples from BM and PB, respectively. Of 53 clinical relapses with MRD results available, 42 were preceded by pos MRD (>0.1%) with a median of 70 (range 20-425) days between first pos MRD and clinical relapse. For the 11 remaining pts, 8 were inadequately sampled with a median time of 189 (82-397) days between last sampling and clinical relapse. One patient had an extramedullary relapse only. Of 31 pts who died without relapse, 19 were consistently MRD neg, while 5 were borderline positive (MRD > 0.1% and <0.5%) during the first 100 days but negative thereafter. Four MRD+ patients died without clinical relapse. Three pts were initially MRD+ but turned negative, all of which had chronic GVHD (cGVHD). Of 136 CCR patients, 94 were consistently MRD neg; 26 were borderline pos (MRD > 0.1% and <0.5%) during the first 100 days followed by neg samples; 16 were MRD positive (either > 0.5% during the first 100d or > 0.1% after 100d) of which 10 had a transition from pos to neg samples (all had cGVHD); one patient was treated for a molecular relapse detected by clinical routine method (FISH) and five patients were MRD positive at time of last follow-up. MRD used as a time-dependent co-variate was negatively associated with RFS (HR 7.1, p<0.01). Estimated cumulative incidence of relapse and non-relapse mortality 2y after pos MRD was 60% and 7% respectively (see figure). Image:Summary/Conclusion: We report the development of a highly functional personalized MRD pipeline based on patient-specific mutations showing a high sensitivity to predict relapse and relapse-free survival.